Substrate for sensors

ABSTRACT

It is an object of the present invention to provide a substrate for sensors and a container, wherein the adhesiveness of the substrate to a thin film is improved, a physiologically active substance can be immobilized without the peeling of the thin film from a plastic substrate, and non-specific adsorption during the analysis of interaction among biomolecules is small. The present invention provides a substrate for sensors, which has a thin film layer on a plastic substrate, wherein the plastic substrate has been treated with an organic primer before formation of the thin film.

TECHNICAL FIELD

The present invention relates to a substrate for sensors and a containerusing a plastic substrate having a thin film on the surface thereof. Thepresent invention particularly relates to a substrate for sensors whichis used for a surface plasmon resonance analysis biosensor.

BACKGROUND ART

Recently, a large number of measurements using intermolecularinteractions such as immune responses are being carried out in clinicaltests, etc. However, since conventional methods require complicatedoperations or labeling substances, several techniques are used that arecapable of detecting the change in the binding amount of a testsubstance with high sensitivity without using such labeling substances.Examples of such a technique may include a surface plasmon resonance(SPR) measurement technique, a quartz crystal microbalance (QCM)measurement technique, and a measurement technique of using functionalsurfaces ranging from gold colloid particles to ultra-fine particles.The SPR measurement technique is a method of measuring changes in therefractive index near an organic functional film attached to the metalfilm of a chip by measuring a peak shift in the wavelength of reflectedlight, or changes in amounts of reflected light in a certain wavelength,so as to detect adsorption and desorption occurring near the surface.The QCM measurement technique is a technique of detecting adsorbed ordesorbed mass at the ng level, using a change in frequency of a crystaldue to adsorption or desorption of a substance on gold electrodes of aquartz crystal (device). In addition, the ultra-fine particle surface(nm level) of gold is functionalized, and physiologically activesubstances are immobilized thereon. Thus, a reaction to recognizespecificity among physiologically active substances is carried out,thereby detecting a substance associated with a living organism fromsedimentation of gold fine particles or sequences.

In all of the above-described techniques, the surface where aphysiologically active substance is immobilized is important. Surfaceplasmon resonance (SPR), which is most commonly used in this technicalfield, will be described below as an example.

A commonly used measurement chip comprises a transparent substrate(e.g., glass), an evaporated metal film, and a thin film having thereona functional group capable of immobilizing a physiologically activesubstance. The measurement chip immobilizes the physiologically activesubstance on the metal surface via the functional group. A specificbinding reaction between the physiological active substance and a testsubstance is measured, so as to analyze an interaction betweenbiomolecules.

As a thin film having a functional group capable of immobilizing aphysiologically active substance, there has been reported a measurementchip where a physiologically active substance is immobilized by using afunctional group binding to metal, a linker with a chain length of 10 ormore atoms, and a compound having a functional group capable of bindingto the physiologically active substance (Japanese Patent No. 2815120).Moreover, a measurement chip comprising a metal film and aplasma-polymerized film formed on the metal film has been reported(Japanese Patent Laid-Open No. 9-264843).

On the other hand, when a thin film is formed on a plastic substrate,and a physiologically active substance is then immobilized on thesurface of the thin film, such a treatment has been problematic in thatadhesive strength between the plastic substrate and the thin filmbecomes insufficient, in that the thin film is peeled from thesubstrate, and thus in that non-specific adsorption of biomolecules ontothe substrate is likely to generate during the production of a sensorand the analysis of interaction among the biomolecules. In particular,the above treatment has been problematic in that if the thin film isallowed to come into contact with an aqueous solution or an organicsolvent, it often causes trouble.

DISCLOSURE OF THE INVENTION

It is an object of the present invention to solve the aforementionedproblem. That is to say, it is an object of the present invention toprovide a substrate for sensors and a container, wherein theadhesiveness of the substrate to a thin film is improved, aphysiologically active substance can be immobilized without the peelingof the thin film from a plastic substrate, and non-specific adsorptionduring the analysis of interaction among biomolecules is small.

As a result of intensive studies directed towards achieving theaforementioned object, the present inventors have found that theaforementioned object can be achieved by subjecting a thin film surface,which has been formed after treating a plastic substrate surface with anorganic primer, to a treatment for immobilizing a physiologically activesubstance, thereby completing the present invention.

The present invention provides a substrate for sensors, which has a thinfilm layer on a plastic substrate, wherein the plastic substrate hasbeen treated with an organic primer before formation of the thin film.

Preferably, the treatment with an organic primer is carried out bycoating the molded plastic substrate with a primer agent.

Preferably, the treatment with an organic primer is carried out bypreviously mixing a primer agent into a plastic material in an amount of0.1% by weight to 10% by weight based on the weight of the plasticmaterial, and then molding the mixture into the plastic substrate.

Preferably, the organic primer is a compound represented by the formula:R¹—X—R² wherein each of R¹ and R² independently represents H orC_(n)H_(2n+1) wherein n represents an integer between 1 and 30; and Xrepresents —C(═O)O—, —O—, —C═O—, —N(R)— wherein R represents a hydrogenatom or a lower alkyl group, or —N(R²)N(R¹)— wherein each of R¹ and R²independently represents a hydrogen atom or a lower alkyl group.

Preferably, n is an integer between 10 and 20.

Preferably, the material of the thin film is a metal or a metal oxide.

Preferably, the material of the thin film consists of a free electronmetal selected from the group consisting of gold, silver, copper,platinum, and aluminum.

Preferably, the substrate for sensors according to the present inventionis a substrate for biosensors.

Preferably, the substrate for sensors according to the present inventionis used in non-electrochemical detection, and is more preferably used insurface plasmon resonance analysis.

Preferably, a linker molecule having a functional group capable ofimmobilizing a physiologically active substance is bound to the surfaceof a vacuum formed film layer via a chemical bond.

Preferably, a polymer film having a functional group capable ofimmobilizing a physiologically active substance is formed on the surfaceof a vacuum formed film layer.

Preferably, the functional group capable of immobilizing aphysiologically active substance is —OH, —SH, —COOH, —NR¹R² (wherein R¹and R² each independently represents a hydrogen atom or lower alkylgroup), —CHO, —NR³NR¹R² (wherein each of R¹, R², and R³ independentlyrepresents a hydrogen atom or lower alkyl group), —NCO, —NCS, an epoxygroup, or a vinyl group.

Preferably, the plastic substrate is substantially transparent.

Preferably, the plastic material that constitutes the substrate is amaterial having a norbornene skeleton.

Preferably, in the substrate for sensors according to the presentinvention, a physiologically active substance is bound to the surfacevia a covalent bond.

In another aspect, the present invention provides a container having athin film layer on a plastic substrate, wherein the plastic substratehas been treated with an organic primer before formation of the thinfilm.

BEST MODE FOR CARRYING OUT THE INVENTION

The embodiments of the present invention will be described below.

The substrate of the present invention is a substrate having a vacuumformed film layer on a plastic substrate treated with an organic primer,which is characterized in that it is allowed to come into contact with aliquid after the vacuum forming of a film.

The term “organic primer” is used in the present invention to mean anorganic substance for improving the adhesiveness of the surface of aplastic substrate to a vacuum formed thin film. An example of a resinorganic primer is an epoxy/phenol resin, a polyamide resin, and amixture of a resorcinol resin and a rubber latex. In addition, a lowmolecular weight compound can also be used as an organic primer. Whenthe organic primer is such a low molecular weight compound, it ispossible not only to apply it to a plastic substrate, but also to mix itwith a plastic material before molding and then to mold the obtainedmixture, thereby forming a primer layer on the surface of the substrate.A preferred low molecular weight organic primer agent can be representedby the formula R¹—X—R².

Herein, each of R¹ and R² independently represents H or C_(n)H_(2n+1)wherein n represents an integer between 1 and 30; and X represents—C(═O)O—, —O—, —C═O—, —N(R)— (wherein R represents a hydrogen atom or alower alkyl group), or —N(R²)N(R¹)— (wherein each of R¹ and R²independently represents a hydrogen atom or a lower alkyl group).

The term “lower alkyl group” is generally used in the presentspecification to mean an alkyl group containing approximately 1 to 10carbon atoms, preferably an alkyl group containing 1 to 8 carbon atoms,and more preferably an alkyl group containing 1 to 6 carbon atoms.Particularly preferably, each of R¹ and R² independently represents H orC_(n)H_(2n+1) wherein n represents an integer between 1 and 20. In thecase of a carbon chain, it is preferably constituted with only a linearstructure having no branches.

Since a carbon chain constituting an organic primer has a linearstructure, even if stress is given between a plastic substrate and athin film, exfoliation can be alleviated. Preferred examples of anorganic primer material may include: higher fatty acid alcohols such aspalmityl alcohol or stearyl alcohol; higher fatty acids such as stearicacid or 12-hydroxy stearic acid; and higher fatty acid esters such asn-butyl myristate.

As a primer treatment method, a coating method can be used. Such coatingcan be carried out according to common methods. Examples of such acoating method may include spin coating, air-knife coating, bar coating,blade coating, slide coating, curtain coating, spray method, evaporationmethod, casting method, and immersion method. As a preferred organicprimer treatment of a substrate surface, a primer agent has previouslybeen mixed into a plastic material in an amount between 0.1% by weightand 10% by weight based on the weight of the plastic material, and themixture is then molded into a plastic substrate. In this case, if theamount of a primer agent is too small, the adhesive strength between theplastic substrate and a vacuum formed film layer is decreased. Incontrast, if the amount of a primer agent is too large, it causesproblems such as a decrease in the optical properties or strength of theplastic substrate.

Preferably, the plastic substrate is substantially transparent, becauseit can be non-electrochemically detected. A preferred plastic materialis a material having low hygroscopicity, low water-absorbing properties,and high transparency. Specifically, a material having a norborneneskeleton is preferred.

Preferred examples of such a plastic substrate used herein may includematerials that are transparent to laser light, such as polymethylmethacrylate, polyethylene terephthalate, polycarbonate, or acycloolefin polymer. Such a substrate is preferably made from amaterial, which does not exhibit anisotropy to polarized light and isexcellent in terms of processability. Such a material is particularlypreferably a hydrocarbon polymer having a norbornene skeleton.

A thin film may be formed by common methods. Examples of such a commonmethod may include sputtering method, evaporation method, ion-platingmethod, and plating method. A thin film material is selected from amonga metal, a metal oxide, a semiconductor, and an organic substance. Thethin film material is preferably a metal or a metal oxide. It is morepreferably a free-electron metal selected from the group consisting ofgold, silver, copper, platinum, and aluminum. The metal is notparticularly limited, as long as surface plasmon resonance is generatedwhen the metal is used for a surface plasmon resonance biosensor.Examples of a preferred metal may include free-electron metals such asgold, silver, copper, aluminum or platinum. Of these, gold isparticularly preferable. These metals can be used singly or incombination. Moreover, considering adherability to the above substrate,an interstitial layer consisting of chrome or the like may be providedbetween the substrate and a metal layer.

The film thickness of a metal film is not limited. When the metal filmis used for a surface plasmon resonance biosensor, the thickness ispreferably between 0.1 nm and 500 nm, and particularly preferablybetween 1 nm and 200 nm. If the thickness exceeds 500 nm, the surfaceplasmon phenomenon of a medium cannot be sufficiently detected.Moreover, when an interstitial layer consisting of chrome or the like isprovided, the thickness of the interstitial layer is preferably between0.1 nm and 10 nm.

After formation of a thin film, the surface of a substrate is chemicallymodified, so that it can immobilize a physiologically active substancethereon. Thereby, interaction among biomolecules is converted to signalssuch as electric signals, and as a result, it becomes possible tomeasure or detect a substance as a target. Thus, a chemical modificationis useful. As the chemical modification, a functional group capable offorming a covalent bond with a physiologically active substance can beintroduced into the surface of a substrate in an aqueous solution or inan organic solvent according to common methods.

Preferred functional group capable of forming a covalent bond with aphysiologically active substance includes —OH, —SH, —COOH, —NR¹R²(wherein each of R¹ and R² independently represents a hydrogen atom orlower alkyl group), —CHO, —NR³NR¹R² (wherein each of R¹, R²and R³independently represents a hydrogen atom or lower alkyl group), —NCO,—NCS, an epoxy group, or a vinyl group. The number of carbon atomscontained in the lower alkyl group is not particularly limited herein.However, it is generally about C1 to C10, and preferably C1 to C6.

Examples of a linker molecule preferably used in the present inventionmay include: proteins such as albumin or casein; sugar derivatives suchas agar, sodium alginate, or a starch derivative; cellulose compoundssuch as carboxymethyl cellulose or hydroxymethyl cellulose;polysaccharides such as chitin or chitosan; and synthetic hydrophilicpolymers such as polyvinyl alcohol, poly-N-vinylpyrrolidone,polyacrylamide, or polyacrylic acid. A hydrophilic polymer compound canbe applied to a substrate according to common coating methods. Examplesof such a common coating method may include spin coating, air-knifecoating, bar coating, blade coating, slide coating, curtain coating,spray method, evaporation method, casting method, and immersion method.

A polymer film material preferably used in the present invention mayinclude polystyrene, polyethylene, polypropylene, polyethyleneterephthalate, polyvinyl chloride, polymethyl methacrylate, polyester,and nylon. These polymer materials can be subjected to a surfacetreatment, such as a chemical treatment using chemicals, couplingagents, surfactants, surface evaporation, etc., or a physical treatmentusing heat, ultraviolet ray, radioactive ray, plasma, ions, etc.

In order to introduce these functional groups into the surface, a methodis applied that involves applying a hydrophobic polymer containing aprecursor of such a functional group on a metal surface or metal film,and then generating the functional group from the precursor located onthe outermost surface by chemical treatment. For example, polymethylmethacrylate, a hydrophobic polymer containing —COOCH₃ group, is appliedon a metal film, and then the surface comes into contact with an NaOHaqueous solution (1N) at 40° C. for 16 hours, so that a —COOH group isgenerated on the outermost surface. In addition, when a polystyrenecoating layer is subjected to a UV/ozone treatment for example, a —COOHgroup and a —OH group are generated on the outermost surface thereof.

The conventional biosensor is comprised of a receptor site forrecognizing a chemical substance as a detection target and a transducersite for converting a physical change or chemical change generated atthe site into an electric signal. In a living body, there existsubstances having an affinity with each other, such as enzyme/substrate,enzyme/coenzyme, antigen/antibody, or hormone/receptor. The biosensoroperates on the principle that a substance having an affinity withanother substance, as described above, is immobilized on a substrate tobe used as a molecule-recognizing substance, so that the correspondingsubstance can be selectively measured.

A physiologically active substance is covalently bound to theabove-obtained substrate for sensor via the above functional group, sothat the physiologically active substance can be immobilized on themetal surface or metal film.

A physiologically active substance immobilized on the substrate forsensor of the present invention is not particularly limited, as long asit interacts with a measurement target. Examples of such a substance mayinclude an immune protein, an enzyme, a microorganism, nucleic acid, alow molecular weight organic compound, a nonimmune protein, animmunoglobulin-binding protein, a sugar-binding protein, a sugar chainrecognizing sugar, fatty acid or fatty acid ester, and polypeptide oroligopeptide having a ligand-binding ability.

Examples of an immune protein may include an antibody whose antigen is ameasurement target, and a hapten. Examples of such an antibody mayinclude various immunoglobulins such as IgG, IgM, IgA, IgE or IgD. Morespecifically, when a measurement target is human serum albumin, ananti-human serum albumin antibody can be used as an antibody. When anantigen is an agricultural chemical, pesticide, methicillin-resistantStaphylococcus aureus, antibiotic, narcotic drug, cocaine, heroin, crackor the like, there can be used, for example, an anti-atrazine antibody,anti-kanamycin antibody, anti-metamphetamine antibody, or antibodiesagainst O antigens 26, 86, 55, 111 and 157 among enteropathogenicEscherichia coli.

An enzyme used as a physiologically active substance herein is notparticularly limited, as long as it exhibits an activity to ameasurement target or substance metabolized from the measurement target.Various enzymes such as oxidoreductase, hydrolase, isomerase, lyase orsynthetase can be used. More specifically, when a measurement target isglucose, glucose oxidase is used, and when a measurement target ischolesterol, cholesterol oxidase is used. Moreover, when a measurementtarget is an agricultural chemical, pesticide, methicillin-resistantStaphylococcus aureus, antibiotic, narcotic drug, cocaine, heroin, crackor the like, enzymes such as acetylcholine esterase, catecholamineesterase, noradrenalin esterase or dopamine esterase, which show aspecific reaction with a substance metabolized from the abovemeasurement target, can be used.

A microorganism used as a physiologically active substance herein is notparticularly limited, and various microorganisms such as Escherichiacoli can be used.

As nucleic acid, those complementarily hybridizing with nucleic acid asa measurement target can be used. Either DNA (including cDNA) or RNA canbe used as nucleic acid. The type of DNA is not particularly limited,and any of native DNA, recombinant DNA produced by gene recombinationand chemically synthesized DNA may be used.

As a low molecular weight organic compound, any given compound that canbe synthesized by a common method of synthesizing an organic compoundcan be used.

A nonimmune protein used herein is not particularly limited, andexamples of such a nonimmune protein may include avidin (streptoavidin),biotin, and a receptor.

Examples of an immunoglobulin-binding protein used herein may includeprotein A, protein G, and a rheumatoid factor (RF).

As a sugar-binding protein, for example, lectin is used.

Examples of fatty acid or fatty acid ester may include stearic acid,arachidic acid, behenic acid, ethyl stearate, ethyl arachidate, andethyl behenate.

When the physiologically active substance is a protein such as antibodyor enzyme, or DNA, the immobilization thereof can be carried out bycovalently binding it to the functional group on the metal surface usingan amino group, a thiol group or the like of the physiologically activesubstance.

A biosensor to which a physiologically active substance is immobilizedas described above can be used to detect and/or measure a substancewhich interacts with the physiologically active substance.

Thus, the present invention provides a method for detecting or measuringa substance interacting with a physiologically active substance which isimmobilized on the biosensor of the present invention, wherein thebiosensor of the present invention to which the physiologically activesubstance is immobilized is used, and a test substance is allowed tocome into contact with said biosensor.

As the test substance, a sample containing a substance interacting withthe aforementioned physiologically active substance, or the like, can beused.

A preferred use of the substrate for sensors according to the presentinvention is a substrate for sensors and a container, which are allowedto come into contact with an aqueous solution or an organic solvent. Aliquid to be allowed to come into contact with the substrate preferablyhas pH of 8 or greater because of the surface modification of a vacuumformed film layer and immobilization of a physiologically activesubstance. For the purpose of realizing dehydration and deacidification,such a liquid to be allowed to come into contact with the substrate morepreferably has pH of 10 or greater.

The form of a container used in the present invention is notparticularly limited, as long as it is able to contain a liquid (forexample, a liquid that contains a physiologically active substance suchas a protein or an agent such as a low molecular weight compound).Examples of the container may include a tube and a plate (e.g. 96-wellplate, etc.).

In the present invention, the interaction of a physiologically activesubstance immobilized on the biosensor surface with a test substance ispreferably detected and/or measured by a non-electrochemical method.Examples of such a non-electrochemical method may include the surfaceplasmon resonance (SPR) measurement technique, the quartz crystalmicrobalance (QCM) measurement technique, and a measurement techniqueusing a functionalized surface ranging from colloidal gold particles toultra-fine particles.

In a preferred embodiment of the present invention, the biosensor of thepresent invention can be used as a biosensor for surface plasmonresonance which is characterized in that it comprises a metal filmplaced on a transparent substrate.

A biosensor for surface plasmon resonance is a biosensor used for asurface plasmon resonance biosensor, meaning a member comprising aportion for transmitting and reflecting light emitted from the sensorand a portion for immobilizing a physiologically active substance. Itmay be fixed to the main body of the sensor or may be detachable.

The surface plasmon resonance phenomenon occurs due to the fact that theintensity of monochromatic light reflected from the border between anoptically transparent substance such as glass and a metal thin filmlayer depends on the refractive index of a sample located on theoutgoing side of the metal. Accordingly, the sample can be analyzed bymeasuring the intensity of reflected monochromatic light.

A device using a system known as the Kretschmann configuration is anexample of a surface plasmon measurement device for analyzing theproperties of a substance to be measured using a phenomenon whereby asurface plasmon is excited with a lightwave (for example, JapanesePatent Laid-Open No. 6-167443). The surface plasmon measurement deviceusing the above system basically comprises a dielectric block formed ina prism state, a metal film that is formed on a face of the dielectricblock and comes into contact with a measured substance such as a samplesolution, a light source for generating a light beam, an optical systemfor allowing the above light beam to enter the dielectric block atvarious angles so that total reflection conditions can be obtained atthe interface between the dielectric block and the metal film, and alight-detecting means for detecting the state of surface plasmonresonance, that is, the state of attenuated total reflection, bymeasuring the intensity of the light beam totally reflected at the aboveinterface.

In order to achieve various incident angles as described above, arelatively thin light beam may be caused to enter the above interfacewhile changing an incident angle. Otherwise, a relatively thick lightbeam may be caused to enter the above interface in a state of convergentlight or divergent light, so that the light beam contains componentsthat have entered therein at various angles. In the former case, thelight beam whose reflection angle changes depending on the change of theincident angle of the entered light beam can be detected with a smallphotodetector moving in synchronization with the change of the abovereflection angle, or it can also be detected with an area sensorextending along the direction in which the reflection angle is changed.In the latter case, the light beam can be detected with an area sensorextending to a direction capable of receiving all the light beamsreflected at various reflection angles.

With regard to a surface plasmon measurement device with the abovestructure, if a light beam is allowed to enter the metal film at aspecific incident angle greater than or equal to a total reflectionangle, then an evanescent wave having an electric distribution appearsin a measured substance that is in contact with the metal film, and asurface plasmon is excited by this evanescent wave at the interfacebetween the metal film and the measured substance. When the wave vectorof the evanescent light is the same as that of a surface plasmon andthus their wave numbers match, they are in a resonance state, and lightenergy transfers to the surface plasmon. Accordingly, the intensity oftotally reflected light is sharply decreased at the interface betweenthe dielectric block and the metal film. This decrease in lightintensity is generally detected as a dark line by the abovelight-detecting means. The above resonance takes place only when theincident beam is p-polarized light. Accordingly, it is necessary to setthe light beam in advance such that it enters as p-polarized light.

If the wave number of a surface plasmon is determined from an incidentangle causing the attenuated total reflection (ATR), that is, anattenuated total reflection angle (θSP), the dielectric constant of ameasured substance can be determined. As described in Japanese PatentLaid-Open No. 11-326194, a light-detecting means in the form of an arrayis considered to be used for the above type of surface plasmonmeasurement device in order to measure the attenuated total reflectionangle (θSP) with high precision and in a large dynamic range. Thislight-detecting means comprises multiple photo acceptance units that arearranged in a certain direction, that is, a direction in which differentphoto acceptance units receive the components of light beams that aretotally reflected at various reflection angles at the above interface.

In the above case, there is established a differentiating means fordifferentiating a photodetection signal outputted from each photoacceptance unit in the above array-form light-detecting means withregard to the direction in which the photo acceptance unit is arranged.An attenuated total reflection angle (θSP) is then specified based onthe derivative value outputted from the differentiating means, so thatproperties associated with the refractive index of a measured substanceare determined in many cases.

In addition, a leaking mode measurement device described in “BunkoKenkyu (Spectral Studies)” Vol. 47, No. 1 (1998), pp. 21 to 23 and 26 to27 has also been known as an example of measurement devices similar tothe above-described device using attenuated total reflection (ATR). Thisleaking mode measurement device basically comprises a dielectric blockformed in a prism state, a clad layer that is formed on a face of thedielectric block, a light wave guide layer that is formed on the cladlayer and comes into contact with a sample solution, a light source forgenerating a light beam, an optical system for allowing the above lightbeam to enter the dielectric block at various angles so that totalreflection conditions can be obtained at the interface between thedielectric block and the clad layer, and a light-detecting means fordetecting the excitation state of waveguide mode, that is, the state ofattenuated total reflection, by measuring the intensity of the lightbeam totally reflected at the above interface.

In the leaking mode measurement device with the above structure, if alight beam is caused to enter the clad layer via the dielectric block atan incident angle greater than or equal to a total reflection angle,only light having a specific wave number that has entered at a specificincident angle is transmitted in a waveguide mode into the light waveguide layer, after the light beam has penetrated the clad layer. Thus,when the waveguide mode is excited, almost all forms of incident lightare taken into the light wave guide layer, and thereby the state ofattenuated total reflection occurs, in which the intensity of thetotally reflected light is sharply decreased at the above interface.Since the wave number of a waveguide light depends on the refractiveindex of a measured substance placed on the light wave guide layer, therefractive index of the measurement substance or the properties of themeasured substance associated therewith can be analyzed by determiningthe above specific incident angle causing the attenuated totalreflection.

In this leaking mode measurement device also, the above-describedarray-form light-detecting means can be used to detect the position of adark line generated in a reflected light due to attenuated totalreflection. In addition, the above-described differentiating means canalso be applied in combination with the above means.

The above-described surface plasmon measurement device or leaking modemeasurement device may be used in random screening to discover aspecific substance binding to a desired sensing substance in the fieldof research for development of new drugs or the like. In this case, asensing substance is immobilized as the above-described measuredsubstance on the above thin film layer (which is a metal film in thecase of a surface plasmon measurement device, and is a clad layer and alight guide wave layer in the case of a leaking mode measurementdevice), and a sample solution obtained by dissolving various types oftest substance in a solvent is added to the sensing substance.Thereafter, the above-described attenuated total reflection angle (θSP)is measured periodically when a certain period of time has elapsed.

If the test substance contained in the sample solution is bound to thesensing substance, the refractive index of the sensing substance ischanged by this binding over time. Accordingly, the above attenuatedtotal reflection angle (θSP) is measured periodically after the elapseof a certain time, and it is determined whether or not a change hasoccurred in the above attenuated total reflection angle (θSP), so that abinding state between the test substance and the sensing substance ismeasured. Based on the results, it can be determined whether or not thetest substance is a specific substance binding to the sensing substance.Examples of such a combination between a specific substance and asensing substance may include an antigen and an antibody, and anantibody and an antibody. More specifically, a rabbit anti-human IgGantibody is immobilized as a sensing substance on the surface of a thinfilm layer, and a human IgG antibody is used as a specific substance.

It is to be noted that in order to measure a binding state between atest substance and a sensing substance, it is not always necessary todetect the angle itself of an attenuated total reflection angle (θSP).For example, a sample solution may be added to a sensing substance, andthe amount of an attenuated total reflection angle (θSP) changed therebymay be measured, so that the binding state can be measured based on themagnitude by which the angle has changed. When the above-describedarray-form light-detecting means and differentiating means are appliedto a measurement device using attenuated total reflection, the amount bywhich a derivative value has changed reflects the amount by which theattenuated total reflection angle (θSP) has changed. Accordingly, basedon the amount by which the derivative value has changed, a binding statebetween a sensing substance and a test substance can be measured(Japanese Patent Application No. 2000-398309 filed by the presentapplicant). In a measuring method and a measurement device using suchattenuated total reflection, a sample solution consisting of a solventand a test substance is added dropwise to a cup- or petri dish-shapedmeasurement chip wherein a sensing substance is immobilized on a thinfilm layer previously formed at the bottom, and then, theabove-described amount by which an attenuated total reflection angle(θSP) has changed is measured.

Moreover, Japanese Patent Laid-Open No. 2001-330560 describes ameasurement device using attenuated total reflection, which involvessuccessively measuring multiple measurement chips mounted on a turntableor the like, so as to measure many samples in a short time.

When the biosensor of the present invention is used in surface plasmonresonance analysis, it can be applied as a part of various surfaceplasmon measurement devices described above.

The present invention will be further specifically described in thefollowing examples. However, the examples are not intended to limit thescope of the present invention.

EXAMPLES Example 1 Substrate (1) of the Present Invention

A Zeonex (manufactured by Zeon Corp.) pellet was melted at 240° C.Thereafter, using an injection molding device, the obtained melt wasmolded into a substrate having a size of 8 mm long×120 mm wide×1.5 mmheight. This substrate was placed in an aluminum container having ahermetically closed structure of 30 mm long×130 mm wide×10 mm deep. Thisaluminum container was fixed on the inner cup of a spin-coater (MODELSC408; manufactured by Nanometric Technology Inc.) equipped with ahermetically closed inner cup, such that the gold surface substrate waspositioned at 135 mm from the center and such that the tangentialdirection of a circular arc became a long axis. 100 μl of an ethanolsolution containing 0.2% 12-hydroxy stearic acid was added dropwise tothe substrate, using a micropipette, so that the entire surface of thegold surface substrate was coated with coating solution A. The aluminumcontainer was hermetically sealed, and it was then left at rest for 30seconds. Thereafter, it was rotated at 200 rpm for 60 seconds. Using aparallel-plate-type 6-inch sputtering apparatus (SH-550; manufactured byUlvac Inc.), a gold film was formed on the substrate by a sputteringtechnique, resulting in a gold thickness of 50 nm, so as to produce thesubstrate (1) of the present invention.

Example 2 Substrate (2) of the Present Invention

The substrate (2) of the present invention was produced by the samemethod as that described in Example 1, with the exception that palmitylalcohol was used instead of 12-hydroxy stearic acid.

Example 3 Substrate (3) of the Present Invention

12-hydroxy stearic acid was added to the Zeonex (manufactured by ZeonCorp.) pellet, in an additive amount of 1% by weight based on the totalweight. Thereafter, the mixture was melted and mixed at 240° C.Thereafter, using an injection molding device, the obtained melt wasmolded into a substrate having a size of 8 mm long×120 mm wide×1.5 mmheight. Using a parallel-plate-type 6-inch sputtering apparatus (SH-550;manufactured by Ulvac Inc.), a gold film was formed on the substrate bya sputtering technique, resulting in a gold thickness of 50 nm, so as toproduce the substrate (3) of the present invention.

Comparative Example Substrate of Comparative Example

The substrate of the comparative example was produced by the same methodas that described in Example 3 with the exception that 12-hydroxystearic acid was not added.

Test Example 1 Evaluation of Gold Film Adhesiveness

With regard to the substrates (1), (2) and (3) of the present inventionand the substrate of the comparative example, 5.0 mM11-hydroxy-1-undecanethiol solution in ethanol/water (80/20) was addedto each substrate in such a way that the solution was allowed come intocontact with the gold film of the substrate, so as to carry out asurface treatment at 25° C. for 18 hours. Thereafter, the resultantsubstrate was washed with ethanol 5 times, then with a mixed solvent ofethanol/water once, and then with water 5 times.

Subsequently, the surface coated with 11-hydroxy-1-undecanethiol wasallowed to come into contact with 10% by weight of an epichlorohydrinsolution (solvent: a mixed solution of 0.4 M sodium hydroxide anddiethylene glycol dimethyl ether at a mixing ratio of 1:1), and thereaction was carried out in a shaking incubator at 25° C. for 4 hours.Thereafter, the surface was washed with ethanol 2 times, and then withwater 5 times.

Subsequently, 4.5 ml of 1 M sodium hydroxide was added to 40.5 ml of anaqueous solution that contained 25% by weight of dextran (T500,Pharmacia), and the obtained solution was then allowed to come intocontact with the epichlorohydrin-treated surface. Thereafter, it wasincubated in a shaking incubator at 25° C. for 20 hours. Thereafter, thesurface was washed with water at 50° C. 10 times. Subsequently, amixture obtained by dissolving 3.5 g of bromoacetic acid in 27 g of 2 Msodium hydroxide solution was allowed to come into contact with theaforementioned dextran-treated surface, and it was then incubated in ashaking incubator at 28° C. for 16 hours. Thereafter, the surface waswashed with water, and the aforementioned procedure was then repeatedonce again. The thus produced substrate is called a dextran-immobilizedsubstrate.

The gold film surface of the produced dextran substrate was observedunder an optical microscope at a magnification of 200 times. In the caseof substrates (1), (2), and (3) of the examples, exfoliation of the goldfilm from the Zeonex substrate was not observed, and thus it was good interms of adhesiveness. On the other hand, in the case of the substrateof the comparative example, exfoliation of 10 gold film portions eachhaving a diameter of approximately 10 μm was observed per area of 1 mm²,and thus it was poor in terms of adhesiveness.

Test Example 2 Evaluation of Non-Specific Adsorption PreventingPerformance

Since non-specific adsorption of proteins on the biosensor surfacecauses noise, such non-specific adsorption preferably occurs at anextremely small degree. Using the dextran-immobilized substrate producedin Test example 1, the non-specific adsorption property of IL8 wasmeasured.

The dextran-immobilized substrate produced in Test example 1 wasinstalled in the device shown in FIG. 22 of Japanese Patent ApplicationLaid-Open No. 2001-330560 (hereinafter referred to as the surfaceplasmon resonance device of the present invention). An HBS-N buffer(manufactured by Biacore) was added to the substrate, and it was thenleft at rest for 20 minutes. Thereafter, an IL8 solution (1 mg/ml HBS-Nbuffer) was added to the substrate, and it was then left at rest for 10minutes. It is to be noted that the HBS-N buffer consists of 0.01 mol/lHEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) (pH 7.4) and0.15 mol/l NaCl. Thereafter, the substrate was washed with the HBS-Nbuffer, and the amount of resonance signal (RU value) changed after 3minutes was defined as the non-specific absorption amount of IL8. Themeasurement results are shown in Table 1. From the results shown inTable 1, it could be confirmed by surface plasmon resonance that abiosensor wherein the substrate of the present invention was used, hadan extremely small degree of non-specific adsorption of proteins. TABLE1 Non-specific Sample adsorption of IL8 Example 1 15 RU Example 2 30 RUExample 3 10 RU Comparative example 100 RU

Thus, it was demonstrated that the present invention provides asubstrate, which prevents exfoliation of a thin film from a plasticsubstrate during the production of the substrate and has only a smalldegree of non-specific adsorption during the analysis of interactionamong biomolecules.

EFFECT OF THE INVENTION

According to the present invention, it became possible to provide asubstrate wherein the adhesiveness of the substrate to a gold film isimproved, a physiologically active substance can be immobilized withoutthe peeling of the thin film from a plastic substrate, and non-specificadsorption during the analysis of interaction among biomolecules issmall.

1. A substrate for sensors, which has a thin film layer on a plasticsubstrate, wherein the plastic substrate has been treated with anorganic primer before formation of the thin film.
 2. The substrate forsensors according to claim 1, wherein the treatment with an organicprimer is carried out by coating the molded plastic substrate with aprimer agent.
 3. The substrate for sensors according to claim 1, whereinthe treatment with an organic primer is carried out by previously mixinga primer agent into a plastic material in an amount of 0.1% by weight to10% by weight based on the weight of the plastic material, and thenmolding the mixture into the plastic substrate.
 4. The substrate forsensors according to claim 1, wherein the organic primer is a compoundrepresented by the formula: R¹—X—R² wherein each of R¹ and R²independently represents H or C_(n)H_(2n+1) wherein n represents aninteger between 1 and 30; and X represents —C(═O)O—, —O—, —C═O—, —N(R)—wherein R represents a hydrogen atom or a lower alkyl group, or—N(R²)N(R¹)— wherein each of R¹ and R² independently represents ahydrogen atom or a lower alkyl group.
 5. The substrate for sensorsaccording to claim 4, wherein n is an integer between 10 and
 20. 6. Thesubstrate for sensors according to claim 1, wherein the material of thethin film is a metal or a metal oxide.
 7. The substrate for sensorsaccording to claim 6, wherein the material of the thin film consists ofa free electron metal selected from the group consisting of gold,silver, copper, platinum, and aluminum.
 8. The substrate for sensorsaccording to claim 1, which is a substrate for biosensors.
 9. Thesubstrate for sensors according to claim 1, which is used innon-electrochemical detection.
 10. The substrate for sensors accordingto claim 1, which is used in surface plasmon resonance analysis.
 11. Thesubstrate for sensors according to claim 1, wherein a linker moleculehaving a functional group capable of immobilizing a physiologicallyactive substance is bound to the surface of a vacuum formed film layervia a chemical bond.
 12. The substrate for sensors according to claim 1,wherein a polymer film having a functional group capable of immobilizinga physiologically active substance is formed on the surface of a vacuumformed film layer.
 13. The substrate for sensors according to claim 11,wherein the functional group capable of immobilizing a physiologicallyactive substance is —OH, —SH, —COOH, —NR¹R² (wherein R¹ and R² eachindependently represents a hydrogen atom or lower alkyl group), —CHO,—NR³NR¹R² (wherein each of R¹, R², and R³ independently represents ahydrogen atom or lower alkyl group), —NCO, —NCS, an epoxy group, or avinyl group.
 14. The substrate for sensors according to claim 12,wherein the functional group capable of immobilizing a physiologicallyactive substance is —OH, —SH, —COOH, —NR¹R² (wherein R¹ and R² eachindependently represents a hydrogen atom or lower alkyl group), —CHO,—NR³NR¹R² (wherein each of R¹, R², and R³ independently represents ahydrogen atom or lower alkyl group), —NCO, —NCS, an epoxy group, or avinyl group.
 15. The substrate for sensors according to claim 1, whereinthe plastic substrate is substantially transparent.
 16. The substratefor sensors according to claim 1, wherein the plastic material thatconstitutes the substrate is a material having a norbornene skeleton.17. The substrate for sensors according to claim 1, wherein aphysiologically active substance is bound to the surface via a covalentbond.
 18. A container having a thin film layer on a plastic substrate,wherein the plastic substrate has been treated with an organic primerbefore formation of the thin film.